Proteolysis of Pseudomonas exotoxin A within hepatic endosomes by cathepsins B and D produces fragments displaying in vitro ADP-ribosylating and apoptotic effects ´ Tatiana El Hage1,2, Severine Lorin3, Paulette Decottignies4,5, Mojgan Djavaheri-Mergny6 and Francois Authier1,2 ¸ ˆ INSERM, Chatenay-Malabry, France ˆtenay-Malabry, France ´ ´ Universite Paris-Sud, Faculte de Pharmacie, Cha ˆ ´ ´ JE 2493, Universite Paris-Sud, Faculte de Pharmacie, Chatenay-Malabry, France CNRS, UMR 8619, Orsay, France ´ Universite Paris-Sud, Orsay, France ´ INSERM VINCO U916, Institut Bergonie, Bordeaux, France Keywords cathepsin; endocytosis; endosome; Pseudomonas exotoxin A; translocation Correspondence ´ F Authier, INSERM, Universite Paris-Sud, ´ Faculte de Pharmacie, rue Jean-Baptiste ˆ ´ Clement, 92296 Chatenay-Malabry, France Fax: +33 46835844 Tel: +33 46835291 E-mail: francois.authier@u-psud.fr (Received 21 March 2010, revised June 2010, accepted 12 July 2010) doi:10.1111/j.1742-4658.2010.07775.x To assess Pseudomonas exotoxin A (ETA) compartmentalization, processing and cytotoxicity in vivo, we have studied the fate of internalized ETA with the use of the in vivo rodent liver model following toxin administration, cell-free hepatic endosomes, and pure in vitro protease assays ETA taken up into rat liver in vivo was rapidly associated with plasma membranes (5–30 min), internalized within endosomes (15–60 min), and later translocated into the cytosolic compartment (30–90 min) Coincident with endocytosis of intact ETA, in vivo association of the catalytic ETA-A subunit and low molecular mass ETA-A fragments was observed in the endosomal apparatus After an in vitro proteolytic assay with an endosomal lysate and pure proteases, the ETA-degrading activity was attributed to the luminal species of endosomal acidic cathepsins B and D, with the major cleavages generated in vitro occurring mainly within domain III of ETA-A Cell-free endosomes preloaded in vivo with ETA intraluminally processed and extraluminally released intact ETA and ETA-A in vitro in a pH-dependent and ATP-dependent manner Rat hepatic cells underwent in vivo intrinsic apoptosis at a late stage of ETA infection, as assessed by the mitochondrial release of cytochrome c, caspase-9 and caspase-3 activation, and DNA fragmentation In an in vitro assay, intact ETA induced ADP-ribosylation of EF-2 and mitochondrial release of cytochrome c, with the former effect being efficiently increased by a cathepsin B ⁄ cathepsin D pretreatment The data show a novel processing pathway for internalized ETA, involving cathepsins B and D, resulting in the production of ETA fragments that may participate in cytotoxicity and mitochondrial dysfunction Abbreviations DT, diphtheria toxin; EEA1, early endosome antigen-1; EF-2, elongation factor-2; ER, endoplasmic reticulum; ETA, exotoxin A; HA, hexa-D-arginine; LRP1, low-density lipoprotein receptor-related protein 1; PA, pepstatin-A; SD, standard deviation; a2MG, a2-macroglobulin FEBS Journal 277 (2010) 3735–3749 ª 2010 The Authors Journal compilation ª 2010 FEBS 3735 Proteolysis of ETA in rat hepatic endosomes T El Hage et al Introduction Exotoxin A (ETA) is considered to be the most toxic factor secreted by Pseudomonas aeruginosa, a Gramnegative opportunistic pathogen infecting immunocompromised individuals and burn victims [1] ETA is a 613 amino acid A ⁄ B exotoxin that kills cells by inhibition of protein synthesis and programmed cell death [2,3] ETA is secreted as a single polypeptide chain composed of three structural and functional domains: domain Ia (amino acids 1–252), which binds to the (a2MG) ⁄ low-density lipoprotein a2-macroglobulin receptor-related protein (LRP1) receptor present in animal cells [4]; domain II (amino acids 253–364), which contains a furin cleavage site (Arg276-Gln277Pro278-Arg279), the Cys265–Cys287 disulfide bond, and a protein translocating sequence (amino acids 280–313) [5,6]; and domain III (amino acids 400– 613), which contains the ADP-ribosylating enzyme [2] To access and ADP-ribosylate its cellular target, elongation factor-2 (EF-2), ETA must be transported across the cellular membrane and into the cytoplasm This is initiated by cell surface binding of ETA to the a2MG ⁄ LRP1 receptor [4], which is followed by internalization of the toxin–receptor complex to the endosomal apparatus by clathrin-dependent and clathrin-independent mechanisms [7] Two subcellular compartments have been proposed as being physiologically relevant to the mechanism of translocation of internalized ETA into the cytosol The first translocation pathway has been proposed to operate at an early stage of endocytosis from endocytic vesicles [8,9] Thus, significant translocation of ETA across the endosomal membrane of mouse lymphocytes was demonstrated, and required exposure of ETA to low endosomal pH and ATP hydrolysis [10] Other studies have proposed that internalized ETA can be retrogradely transported to the endoplasmic reticulum (ER) for retrotranslocation to the cytosol through the Sec61 complex [11] The ER trafficking pathway of ETA might have multiple routes [7], one being the previously characterized KDEL pathway involving the REDLK C-terminal sequence of the toxin [12] Whatever the pathway enabling cytosolic delivery of ETA, activating processes have been proposed to occur at various stages of ETA trafficking These activating steps include furin-mediated cleavage at the Arg279Glu280 peptide bond [13], reduction of the disulfide bond linking Cys265 and Cys287 [14], and removal of the C-terminal Lys [15] Thus, for full ADP-ribosylation of cytosolic EF-2, it was previously suggested that intracellular production of a 37 kDa C-terminal ETA fragment must occur by the sequential action of a 3736 furin-like protease and an undiscovered reductive enzyme [2,13,16] These observations are consistent with the toxin-resistant phenotype of cells lacking furin, which can be abolished by transfection with a cDNA encoding furin [17] However, although proteolytic and reductive processing of ETA should be required for ETA cytotoxicity through the retrograde transport pathway [18], it has not been clearly determined whether ETA requires proteolytic and ⁄ or reductive processing activation to reach the cytosol through the endosomal pathways and kill cells [19] Hence, recent studies have suggested that ETA cytotoxicity results largely from endosomal translocation of the intact nonproteolyzed and nonreduced polypeptide toxin [19] At present, no in vivo data exist to support a specificity of requirement for ETA processing and reduction according to the translocation pathway used (endosome or ER) Consequently, in the present study, we used the in situ rat liver model system following toxin administration to rats and cell-free hepatic endosomes to relate the endosomal processing of internalized ETA to toxin cytotoxicity in a physiological state Following administration of ETA to rats, rapid endocytosis of the intact unprocessed ETA was observed, coincident with the endosomal association of the ETA-A subunit (fast association) and low molecular mass ETA-A fragments (slow association) Our results assign an important role to endosomal acidic cathepsins B and D in generating ETA fragments displaying high in vitro ADP-ribosyltransferase activity towards cytosolic EF-2 We report on the in vivo association of ETA and ETA-A with cytosolic fractions, and the in vitro ATP-dependent and pH-dependent translocation of ETA and ETA-A from cell-free endosomes into the external milieu Finally, the mitochondrial release of cytochrome c, activation of caspase-9 and caspase-3 and DNA fragmentation were detected in cytosolic fractions isolated h after ETA treatment, relating for the first time activation of the intrinsic apoptotic pathway with ETA cytotoxicity in a physiological state Results In vivo endocytosis and metabolic fate of ETA in rat liver The kinetics of in vivo uptake of ETA at the hepatic cell surface (plasma membranes) (Fig 1A) and intracellularly (endosomes) (Fig 1B) were assessed first Rats were given an intravenous injection of native FEBS Journal 277 (2010) 3735–3749 ª 2010 The Authors Journal compilation ª 2010 FEBS T El Hage et al Proteolysis of ETA in rat hepatic endosomes A B Plasma membranes Nonreducing conditions _ 15 Endosomes 30 60 90 (min, postinjection) Nonreducing conditions _ 15 30 60 90 (min, postinjection) ETA ETA ETA-A ETA-A Reducing conditions (66 kDa) ETA – 100 – 75 Reducing conditions (66 kDa) ETA – 50 (37 kDa) ETA-A – 37 – 100 – 75 – 50 (37 kDa) ETA-A – 25 – 37 – 25 – 15 – 15 kDa kDa Fig Kinetics of appearance of ETA in hepatic plasma membranes and endosomes after toxin administration Rat hepatic plasma membrane (A) and endosomal fractions (B) were isolated at the indicated times after the in vivo administration of native ETA, and evaluated for their content of internalized toxin by nonreducing (upper blots) and reducing SDS ⁄ PAGE (lower blots) followed by western blot analysis with the polyclonal antibody against ETA Each lane contained 10 lg (plasma membranes) or 30 lg (endosomes) of protein The arrows to the left of each panel indicate the mobilities of intact ETA ( 66 kDa), ETA-A ( 37 kDa), and unknown degradation fragments Molecular mass markers are indicated to the right of the reducing blots The antibody against ETA also binds to undefined plasma membrane proteins distinct from ETA under nonreducing conditions [upper blot in (A)] in both control and toxin-injected rats, one of which had a molecular mass identical to that of ETA-A ETA (15 lg per 100 g body weight) and killed 5–90 postinjection Following preparation of hepatic subcellular fractions, the amount of internalized ETA was determined by SDS ⁄ PAGE followed by western blot analyses with antibody directed against ETA-A It was assumed that the in vivo generation of free ETA-A was attributable to both reductive and proteolytic cleavages occurring within the ETA sequence Thus, both processing pathways were analyzed, under either nonreducing (cleavage analysis at the Cys265–Cys287 disulfide bond; upper blots in Fig 1) or reducing (cleavage analysis at peptide bonds; lower blots in Fig 1) conditions ETA association with plasma membranes was rapid (5 postinjection) and maximal at 5–30 postinjection, before decreasing with time (Fig 1A) A transient association of ETA-A with plasma membranes was also observed under reducing and nonreducing conditions at 15–60 postinjection (Fig 1A) As compared with plasma membranes, endosomal association of both ETA and ETA-A was slightly delayed, with the maximum being observed at 15–60 (ETA) or 30–90 (ETA-A) (Fig 1B) Low molecular mass ETA fragments (< 25 kDa) were immunodetected, especially in endosomal fractions under reducing conditions (Fig 1B, lower blot) Although it has been suggested that it is the ETA– ETA receptor complex that is internalized into toxintreated cells, there are no published reports on the fate of the ETA receptor during toxin endocytosis To determine whether the ETA receptor was cointernalized along with the toxin, the in vivo effect of ETA treatment on the a2MG ⁄ LRP1 receptor in the rat liver endosomal fraction was determined by immunoblotting (Fig 2A, upper blot) A high concentration of a membrane-bound 80 kDa fragment of LRP1 containing the tail epitope was found in the endosomal fraction isolated from control rats The extensive fragmentation of LRP1 within hepatic endosomes may explain, in part, the failure to detect intact LRP1 by us (this study) and others [20] In vivo injection of native ETA effected a rapid increase in endosomal truncated LRP1, with maximal accumulation at 5–15 postinjection By 60 postinjection, the 80 kDa LRP1 species had returned to basal levels (Fig 2A, upper blot) However, the level of the endosomal marker early endosome antigen-1 (EEA1) was not modified after ETA treatment (Fig 2A, lower blot) LRP1 enables endocytosis of ETA and various other ligands among such as a2MG [21] To examine the effect of a2MG on the uptake of ETA into hepatic endosomes, a2MG (15 lg per 100 g body weight) was coinjected with ETA into rats (Fig 2B) Endosomal association of intact ETA and ETA-A was reduced by a2MG coinjection We have previously reported that antibodies reacting with the ER-retention KDEL motif are useful in assessing the integrity of the C-terminal region of cholera toxin [22] As it was unknown whether antibodies FEBS Journal 277 (2010) 3735–3749 ª 2010 The Authors Journal compilation ª 2010 FEBS 3737 Proteolysis of ETA in rat hepatic endosomes A T El Hage et al ETA _ 15 30 60 90 (min, postinjection) Arbitrary units α-LRP1 (tail) LRP1 (80 kDa) 200 * * 100 _ 15 30 60 90 (min, postinjection) ETA _ 15 30 60 90 (min, postinjection) α-EEA1 EEA1 (180 kDa) ETA B 15 ETA/a2MG 30 15 30 (min, postinjection) – 100 – 75 ETA (66 kDa) – 50 ETA-A (37 kDa) – 37 – 25 – 15 kDa C _ Furin _ Dithiothreitol + + ETA _ 15 30 60 90 (min, postinjection) ETA (66 kDa) ETA-A (37 kDa) ETA-A (37 kDa) α-KX5KDEL α-KX5KDEL against KDEL bind to the ETA C-terminal sequence REDLK (which resembles the ER motif KDEL), we first characterized antibodies against KDEL for their binding to native ETA and ETA-A by western blot analysis (Fig 2C, left and middle blots) One antibody, anti-KX5KDEL, bound to ETA-A but not to native ETA (Fig 2C, left and middle blots), whereas the others, anti-KSEKKDEL and anti-KAVKKDEL, did not show any immunoreactivity (results not shown) Therefore, the antibody against KX5KDEL was used to assess the integrity of the REDLK peptide in endosomal ETA-A under reducing conditions (Fig 2C, right blot) KDEL immunoreactivity to internalized ETA-A was detected in endosomal fractions isolated 3738 Fig Characterization of ETA endocytosis into the endosomal apparatus (A) Changes in LRP1 concentration in the endosomal fraction following ETA injection into rats Hepatic endosomal fractions were isolated at the indicated times after the in vivo administration of native ETA, and evaluated for their content of LRP1 (upper blot) or EEA1 (lower blot) by reducing SDS ⁄ PAGE followed by western blot analysis Each lane contained 30 lg (a-LRP1 blot) or 50 lg (a-EEA1 blot) of endosomal protein The LRP1 bands were quantified by scanning densitometry, and the signal intensities for the ETA-treated rats were expressed as a percentage (mean ± SD) of the signal intensity for the control rats (lane )) *P < 0.05 for the differences between ETA ⁄ or ETA ⁄ 15 and control rats ()) The arrows to the right indicate the mobilities of membranebound LRP1 fragment ( 80 kDa) or EEA1 ( 180 kDa) Uncleaved LRP1 ( 600 kDa) was not observed in endosomal fractions from control and toxin-injected rats (B) Effect of a2MG treatment on the internalization of ETA Rat hepatic endosomal fractions were isolated at the indicated times after the in vivo coadministration of ETA and a2MG (15 lg per 100 g body weight), and evaluated for their content of internalized toxin by reducing SDS ⁄ PAGE followed by western blot analysis with the polyclonal antibody against ETA Each lane contained 50 lg of endosomal protein The arrows to the left indicate the mobilities of intact ETA ( 66 kDa), ETA-A ( 37 kDa), and unknown degradation fragments Molecular mass markers are indicated to the right (C) Assessment of immunoreactivity of antibody against KDEL for native and internalized ETA ETA was either untreated (left blot, lane )) or digested in vitro with 100 mL)1Ỉmg)1 furin and 10 mM dithiothreitol (middle blot, lane +), and samples were then analyzed by reducing SDS ⁄ PAGE followed by western blotting with polyclonal antiserum against the synthetic peptide KX5KDEL ETA-A was detected under the latter experimental conditions Rat liver endosomal fractions were then isolated at the indicated times after the in vivo administration of ETA, and evaluated by western blotting for their immunoreactivity with polyclonal antibody against KX5KDEL (blot on the right) [22] The antibody against KX5KDEL also binds to undefined endosomal proteins distinct from ETA, both in control and in toxin-injected rats, whose levels have been shown to be modified by toxin treatment [22] Each lane contained 80 lg of endosomal protein The mobilities of intact ETA ( 66 kDa) and ETA-A ( 37 kDa) are indicated from the livers of rats killed at 15–90 postinjection, with kinetics similar to those of ETA-A uptake into endocytic components (Fig 1B), suggesting that the C-terminal motif REDLK might not be completely removed from ETA-A within the endosomal apparatus Endosomal proteolysis of internalized ETA by cathepsins B and D To confirm the endosomal proteolysis of internalized ETA under conditions that maintained endosome integrity, we used cell-free endosomes containing in vivo internalized ETA (Fig 3A,B) Endosomes were isolated 30 following ETA injection, and intact endocytic vesicles were incubated for various times at FEBS Journal 277 (2010) 3735–3749 ª 2010 The Authors Journal compilation ª 2010 FEBS T El Hage et al Proteolysis of ETA in rat hepatic endosomes pH A 15 pH + ATP 30 15 30 (min) 100 – 75 – ETA (66 kDa) 50 – 37 – ETA-A (37 kDa) 25 – 15 – kDa Arbitrary units ETA ETA-A subunit 150 100 50 _ + 30 30 _ + B 30 30 (min) _ + (ATP) _ _ PMSF PA EDTA HA E64 pH + ATP 60 60 60 60 60 60 (min) (Inhibitor) (66 kDa) ETA (37 kDa) ETA-A C ETA + Cath-D _ ETA + Cath-B 4 5 4 5 6 15 60 15 60 15 60 15 60 15 60 15 pH 60 (min) ETA (66 kDa) ETA-A (37 kDa) neutral pH (pH 7) and 37 °C in an isotonic buffer (which mimicked the intracellular milieu) in the presence or absence of ATP, the substrate of the vacuolar H+-ATPase pump (Fig 3A) Immunoblot analyses showed a progressive loss of intact ETA and ETA-A in the presence of ATP, with concomitant generation of ETA and ETA-A fragments Incubation in the Fig Assessment of ETA-degrading activity associated with hepatic endosomes (A) Rat hepatic endosomal fractions were isolated 30 after ETA administration (15 lg per 100 g body weight) and incubated for the indicated times at 37 °C in isotonic buffer containing 0.15 M KCl, 25 mM Hepes (pH 7), mM MgCl2, and mM CaCl2, in the presence or absence of 10 mM ATP The integrity of ETA was then evaluated by reducing SDS ⁄ PAGE followed by western blotting with the polyclonal antibody against ETA Each lane contained 50 lg of endosomal protein The arrows to the right indicate the mobilities of intact ETA ( 66 kDa), ETA-A ( 37 kDa), and unknown degradation fragments Molecular mass markers are indicated to the left ETA and ETA-A signals were quantified by scanning densitometry, and the signal intensities after a 30 incubation were expressed as a percentage (mean ± SD) of initial values (0 min) (lower panel) (B) Rat hepatic endosomal fractions were isolated 30 after ETA administration (15 lg per 100 g body weight) and incubated at 37 °C in isotonic buffer containing 0.15 M KCl, 25 mM Hepes (pH 7), mM MgCl2, mM CaCl2 and 10 mM ATP for the indicated times in the presence or absence (lane )) of mM phenylmethanesulfonyl fluoride (PMSF), 10 lgỈmL)1 PA, mM EDTA, 10 lM HA, or 10 lM E64 The integrity of ETA was then evaluated by reducing SDS ⁄ PAGE followed by western blotting with the polyclonal antibody against ETA Each lane contained 50 lg of endosomal protein The arrows to the left and right indicate the mobilities of intact ETA ( 66 kDa), ETA-A ( 37 kDa), and unknown degradation fragments (C) Native ETA (10 lg) was incubated at 37 °C with cathepsin D (Cath-D) or cathepsin B (Cath-B) (5 mL)1Ỉmg)1) in 50 mM citrate ⁄ phosphate buffer (pH 4–6) or 50 mM Hepes (pH 7) in the presence of 10 mM CaCl2 and 10 mM dithiothreitol for the indicated times The integrity of ETA was then evaluated by reducing SDS ⁄ PAGE followed by western blotting with the polyclonal antibody against ETA The arrows to the left and right indicate the mobilities of intact ETA ( 66 kDa), ETA-A ( 37 kDa), and unknown degradation fragments absence of ATP revealed a small amount of degradation for intact ETA, whereas no degradation was observed for ETA-A (Fig 3A) We next examined the effects of various protease inhibitors on the proteolysis of endosomal ETA and ETA-A, using cell-free endosomes preloaded with ETA toxin in vivo and incubated in vitro at pH in the presence of ATP (Fig 3B) Western blot analysis with the antibody against ETA revealed that the endosomal ETA-degrading activity was partially inhibited by the aspartic acid protease inhibitor pepstatin-A (PA), the cysteine protease inhibitor E64, and the metalloprotease inhibitor EDTA The inhibition of ETA-degrading activity by PA and E64, its low pH optimum and its presence in the endosomal lumen as a soluble form (results not shown) suggested cathepsins B and D as likely candidates for this activity We therefore examined the hydrolysis of ETA by pure cathepsins B and D at pH 4–7 (Fig 3C) FEBS Journal 277 (2010) 3735–3749 ª 2010 The Authors Journal compilation ª 2010 FEBS 3739 Proteolysis of ETA in rat hepatic endosomes T El Hage et al A ETA ETA (66 kDa) kDa – 100 – 75 – 50 – 37 + + + + 4a + Cathepsin B + Cathepsin D pH of medium 4d 4b 6a 6c 6b – 25 6d 4c – 15 B + 0.25 0.25 0.25 Incubation time (h) 1AEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAIDN ETA-B ALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNAGN QLSHMSPIYTIEMGDELLAKLARDATFFVRAHESNEMQPTLAISHAGVSVVMAQTQPRREKR WSEWASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRL HFPEGGSLAALTAHQACHLPLETFTRHRQPR279 280 ETA-A GWEQLEQCGYPVQRLVALYLAARLSWNQVDQV IRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAANADVVSLTCP VAAGECAGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYV FVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGA LLRVYVPRSSLPGFYRTSLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWP LAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLK613 Fig Structural characteristics of ETA fragments generated by cathepsin B and cathepsin D (A) Native ETA (10 lg) was incubated with bovine cathepsin B or cathepsin D (5 mL)1Ỉmg)1), or a mixture of both, at 37 °C for the indicated times in 50 mM citrate ⁄ phosphate buffer (pH 4–6) The incubation mixtures were then analyzed by reducing SDS ⁄ PAGE followed by Coomassie Brilliant Blue Staining The major degradation products generated at pH (peptides 4a–c) or pH (peptides 6a–d) were subjected to N-terminal sequence analysis (B) Sequences of ETA-A and ETA-B The A and B moieties are connected by both a peptide bond (Arg279-Gly280) and a disulfide bridge (Cys265–Cys287) The peptides in red correspond to the N-terminal sequence of intermediates shown in (A): AEEAFDL for intermediates 1, 4a, 4b, 4d, 6a and 6c; CPVAAGECA for intermediates 6b and 6d; and PALA for intermediate 4c Western blot analysis with an antibody against ETA showed that cathepsins B and D degraded ETA in a pH-dependent manner, with maximal degradation being observed at pH The ETA fragments generated by the pure cathepsins (especially cathepsin B at pH 4) had molecular masses very similar to those seen with the endosomal fractions We then assessed the major proteolytic cleavages induced by cathepsin B and ⁄ or D within the ETA sequence at various pH values (Fig 4A,B) The proteolysis of ETA at pH or by cathepsin B and ⁄ or D was analyzed by reducing SDS ⁄ PAGE (Fig 4A), and the cleavage sites in the major metabolites were determined by N-terminal sequence analysis (Fig 4B) Edman degradation of intermediates 4a, 4b, 4d, 6a and 6c revealed the N-terminal sequence of ETA (AEEAFDL), suggesting that the cleavage sites are located within the C-terminal region of the toxin N-terminal sequence analysis of ETA fragments 6b and 6d, generated at pH 6, revealed cleavages between Thr396 and Cys397 (as demonstrated by the CPVAAGECA sequence) For peptide 4c, generated at pH 4, N-terminal sequence analysis revealed the peptide PALA, suggesting cleavage between Asp499 and Pro500 3740 Assessment of cytosolic translocation of internalized ETA We next determined the presence of ETA in cytosolic fractions prepared from ETA-injected rats, using western blot analysis (Fig 5A) The intact 66 kDa ETA toxin was strongly detected within cytosolic fractions at 0.5–4 h postinjection, and lower but detectable immunoreactivity for ETA-A was observed at 1–4 h under both reducing and nonreducing conditions The translocation of endosomal ETA into the extraendosomal milieu was then assessed with intact endosomes isolated 30 after the injection of ETA and incubated for 0–2 h in isotonic medium at 37 °C in the presence or absence of ATP (Fig 5B) Western blot analysis of the ETA associated with sedimentable endosomes showed progressive decreases in immunoreactive ETA and ETA-A at acidic pH (pH 5) or at pH in the presence of ATP Concomitantly, immunoreactive ETA (high level) and ETA-A (low level) were progressively detected in the extraendosomal milieu, confirming the translocation of ETA toxin across the endosomal membrane at acidic luminal pH No ATP-dependent translocation of ETA was observed in the presence of bafilomycin, the H+-ATPase inhibitor FEBS Journal 277 (2010) 3735–3749 ª 2010 The Authors Journal compilation ª 2010 FEBS T El Hage et al Proteolysis of ETA in rat hepatic endosomes A Cytosol _ 0.5 1.5 (h, postinjection) ETA Nonreducing conditions ETA-A ETA (66 kDa) Reducing conditions ETA-A (37 kDa) B Supernatant Pellet (endosomal medium) (extraendosomal medium) _ 0.5 (incubation, h) _ 0.5 (66 kDa) ETA ETA-A (37 kDa) Medium: pH (66 kDa) ETA ETA-A (37 kDa) Medium: pH + ATP (66 kDa) ETA ETA-A (37 kDa) Medium: pH (66 kDa) ETA ETA-A (37 kDa) Medium: pH + ATP + Bafilomycin Fig In vivo and in vitro assessment of the cytosolic translocation of endosomal ETA (A) Rat hepatic cytosolic fractions were isolated at the indicated times after the in vivo administration of native ETA, and evaluated for their toxin content by nonreducing (upper blot) and reducing (lower blot) SDS ⁄ PAGE followed by western blot analysis with the polyclonal antibody against ETA Each lane contained 30 lg of cytosolic protein The arrows to the left indicate the mobilities of intact ETA ( 66 kDa) and ETA-A ( 37 kDa) (B) Membrane translocation of toxin peptides in cell-free rat hepatic endosomes containing in vivo internalized ETA The endosomal fraction was isolated 30 after the administration of ETA, and then resuspended in 0.15 M KCl containing mM MgCl2 and, when indicated, 50 mM Hepes (pH 7), 50 mM citrate ⁄ phosphate buffer (pH 5), 10 mM ATP, and lM bafilomycin After the indicated times of incubation at 37 °C, endosomes were sedimented by ultracentrifugation, and the pellets (endosome-associated material) and supernatants (extraendosomal material) were evaluated for their content of ETA peptides by reducing SDS ⁄ PAGE followed by western blotting with the polyclonal antibody against ETA Equivalent volumes of each subfraction (40 lL) were loaded onto each lane The arrows to the left indicate the mobilities of intact ETA ( 66 kDa) and ETA-A ( 37 kDa) Potential role of cathepsins B and D in the cytotoxic activity of ETA towards cytosolic targets We first examined whether, under conditions where ETA was processed by cathepsins B and D, a corresponding change in the toxin cytotoxicity towards cytosolic EF-2 would be observed (Fig 6A) ETA was first partially processed by a mixture of cathepsins B and D at pH or 6, and then incubated at neutral pH with cytosolic EF-2 in the presence of [32P]NAD A low level of ADP-ribosylation of EF-2 was evident after addition of untreated ETA to the cytosolic fraction After treatment of ETA with cathepsins B and D, EF-2 labeling was increased, especially under acidic conditions (pH > pH > pH 4) However, cathepsin treatment of ETA in the presence of protease inhibitors revealed [32P]NAD-ribose incorporation into cytosolic EF-2 comparable to that observed in the absence of protease treatment A role for mitochondria in ETA-induced cell death has been previously shown with the use of human airway epithelial target cells [23] Consequently, we examined cytochrome c release from cell-free mitochondria isolated from control rats and then treated with ETA in vitro (Fig 6B, upper blots) Cytochrome c association with intact rat liver mitochondria persisted during the incubation in isotonic medium, despite small but detectable release at 15 However, there was substantial release of cytochrome c into the resulting mitochondrial supernatant after the addition of native ETA or ETA that had been pretreated with a mixture of cathepsins B and D No detectable release of cytochrome c was observed following treatment of mitochondria with a mixture of cathepsins B and D alone for the same incubation times (results not shown) To assess the physiological release of mitochondrial cytochrome c into the cytosol, hepatic cytosolic fractions isolated after the in vivo injection of ETA into rats were analyzed for their cytochrome c content by immunoblot analysis (Fig 6B, lower blots) Low but detectable immunoreactivity towards cytochrome c was observed in cytosol isolated from noninjected rats In response to ETA, a strong increase in cytochrome c was observed at h, with the level remaining elevated up to h By contrast, administration of diphtheria toxin (DT) (a toxin that does not access the cytoplasm of rodent cells [24]) did not cause a detectable change in the level of cytochrome c in the cytoplasmic compartment The involvement of caspases in ETA-triggered programmed cell death was then analyzed by incubating hepatic cytosol isolated from ETA-treated rats with fluorescent substrates specific for caspase-9, caspase-3 and caspase-8 (Fig 6C, open bars) Caspase-9 and FEBS Journal 277 (2010) 3735–3749 ª 2010 The Authors Journal compilation ª 2010 FEBS 3741 Proteolysis of ETA in rat hepatic endosomes Cathepsin-treated ETA + cytosol A _ EF-2 EF-2 B Native ETA no toxin WB: α-EF-2 (105 kDa) (ratio of [32P]EF2/EF2) 4+i (pH of proteolysis) ADP-ribosylation of EF-2 (105 kDa) Arbitrary units T El Hage et al _ 15 _ 15 Cathepsin-treated ETA _ 15 (min of incubation) Intact mitochondria 200 * Disrupted mitochondria * 100 Cytochrome c Cytochrome c (15 kDa) (15 kDa) Toxin _ _ 4+i (pH of proteolysis) 0.5 1.5 Cytochrome c ETA C ETA DT (h, postinjection) (15 kDa) Caspase-9 Cytochrome c DT (15 kDa) D Caspase-3 Caspase-8 Cell death (fold stimulation) Caspase activity (fold stimulation) ETA DT 0.5 _ _ 0.5 1.5 15 30 60 90 120 240(min, postinjection) (h, postinjection) Fig Assessment of cytotoxic activity of cathepsin-treated ETA towards cytosolic target and mitochondria (A) Native ETA (10 lg) was digested in vitro for 30 at 37 °C with a mixture of cathepsins B and D (5 mL)1Ỉmg)1) in 25 mM Hepes (pH 7) or 25 mM citrate ⁄ phosphate buffer (pH 4–6) containing 0.1 M dithiothreitol (DT) and, when indicated, lgỈmL)1 PA and lM E64 (lane + i) The treated ETA (1 lg) was then incubated for 15 at 30 °C with the EF-2 associated with the soluble cytosolic fraction (150 lg) in 0.1 M Hepes (pH 7.4) in the presence of lM [32P]NAD Samples (20 lg) were then subjected to SDS ⁄ PAGE and analyzed by autoradiography The dried gels were exposed to X-ray film at )80 °C for 1–3 days The arrow to the left indicates the mobility of 32P-labeled EF-2 ( 105 kDa) Samples (20 lg) were also evaluated for their content of EF-2 using polyclonal antibody against EF-2 The arrow to the left indicates the mobility of EF-2 ( 105 kDa) For each incubation condition, radiolabeled and nonradiolabeled EF-2 signal intensities were quantified by scanning densitometry, and the ratio of [32P]EF-2 signal ⁄ nonradiolabeled EF-2 signal was expressed as a percentage (mean ± SD) of that of untreated ETA (lane ), 100%) (lower panel) *P < 0.05 for the differences between pH or pH and untreated cytosol (B) Upper blots: rat liver mitochondria (7.5 mgỈmL)1) were incubated in either 0.15 M KCl isotonic buffer (intact mitochondria, blot at the top) or hypotonic buffer (disrupted mitochondria, lower blot) in the presence or absence of native or cathepsin-treated ETA After the indicated times, samples were centrifuged and mitochondrial supernatants were carefully separated and mixed with sample buffer Equivalent volumes (30 lL) were subjected to reducing SDS ⁄ PAGE followed by western blot analysis for the in vitro release of cytochrome c The arrows to the right indicate the mobility of cytochrome c ( 15 kDa) Lower blots: rat hepatic cytosolic fractions were isolated at the indicated times after the in vivo administration of native ETA or diphtheria toxin (DT), and evaluated by western blotting with monoclonal antibody for their content of cytochrome c Each lane contained 30 lg of cytosolic protein The arrows to the right indicate the mobility of cytosolic cytochrome c ( 15 kDa) (C) Hepatic cytosolic fractions isolated from ETA-injected or DT-injected rats were incubated with fluorescent substrates specific for caspase-9, caspase-3, and caspase-8 The results are expressed as fold stimulation of fluorescence intensity, normalized to that seen in the control rats, and represent the mean ± SD of three determinations performed on the cytosolic fraction prepared from separate liver fractionations (D) Histone-associated DNA fragments associated with hepatic cytosolic fractions isolated from ETA-injected and DT-injected rats were analyzed by immunoassay Results are expressed as fold stimulation, normalized to that seen in the control rats, and represent the mean of two determinations performed on the cytosolic fractions prepared from separate liver fractionations 3742 FEBS Journal 277 (2010) 3735–3749 ª 2010 The Authors Journal compilation ª 2010 FEBS T El Hage et al caspase-3 activity increased in rat liver cytosol 1.5–2 h after the injection of ETA, with a maximal effect of  2.7-fold (caspase-9) or 4.0-fold (caspase-3) at h No activation of caspase-8 (involved in the extrinsic death receptor pathway) was observed No increase in caspase activity was observed in hepatic cytosolic fractions isolated from DT-injected rats (Fig 6C, closed bars) Finally, the kinetics and extent of production of histone-associated DNA fragments in hepatic cytosolic fractions following ETA administration into rats paralleled caspase-9 and caspase-3 activation, with DNA fragmentation being observed h after ETA injection and remaining elevated up to h (Fig 6D, open bars) No DNA fragmentation was observed in hepatic cytosolic fractions isolated from DT-injected rats (Fig 6D, closed bars) Discussion Using the in situ liver model system, we have previously shown that, after cholera toxin binding to hepatic cells, cholera toxin accumulates in a low-density endosomal compartment and then undergoes endosomal proteolysis by the aspartic acid protease cathepsin D [22,25] Using a similar methodology, others have previously shown that the plant toxin ricin follows a similar intraendosomal processing pathway, requiring ATP-dependent endosomal acidification [26] We have recently extended these observations to DT, and demonstrated the endosomal processing of the internalized toxin in a sequential degradation pathway beginning early, prior to organelle acidification via a neutral furin activity, and followed later under acidic conditions via cathepsin D [24] In the present work, we have evaluated the relationship between the endosomal processes and cytotoxicity of ETA, another A ⁄ B toxin functionally related to DT that has an identical intracellular target (cytosolic EF-2) [6] Our data clearly show that internalized ETA is susceptible to hydrolysis by cathepsins B and D, which are present in hepatic endosomes and operate at acidic pH Comparable to the endosomal degradation of internalized CT [22,25] and ricin [26] in rat hepatic endosomes, the endosomal processing of internalized ETA occurred mainly (if not totally) following ATP-dependent acidification of the endosomal lumen Cytosolic translocation of endosomal ETA was established through the immunodetection of the toxin in cytosol isolated from ETA-injected rats and the use of cell-free endosomes Thus, intact ETA and ETA-A were the only ETA species detected in vivo in the soluble cytosolic fraction after toxin administration and in vitro in the extraendosomal medium during a Proteolysis of ETA in rat hepatic endosomes cell-free translocation assay However, we cannot exclude the possibility that a small number of ETA fragments generated by endosomal cathepsins B and D physiologically translocate from the endosomal lumen to the cytoplasm and interact with cytosolic targets (EF-2 and mitochondria) Low production and ⁄ or translocation of ETA fragments, as well as short halflives in the cytosolic compartment, may well explain why they were not detected Alternatively, the processed fragments may have lost structural elements essential for translocation across the endosomal membrane On the other hand, endosomal proteolysis of ETA may represent a degradative pathway related to the deactivation and termination of intracellular toxin cytotoxicity Clearly, further studies are required to determine whether ETA fragments generated by endosomal cathepsins B and D fully participate in the cytotoxic action of ETA in hepatic tissue Intravenously injected ETA is taken up efficiently by the liver at an early time after death (5 postinjection), suggesting a high binding capacity of ETA in hepatic parenchyma Indeed, injection of the toxin into mice has been shown to result in an early and profound inhibition of hepatic protein synthesis [27] Our results suggest that a2MG ⁄ LRP1 contributes, at least in part, to ETA endocytosis in rat liver in vivo, based on the following: (a) the injection of a2MG, which partially reduced the endosomal association and processing of coinjected ETA; and (b) a time-dependent increase in immunodetectable a2MG ⁄ LRP1 in hepatic endosomes induced by the toxin injection It has been proposed that proteolytic nicking of ETA at the Arg279-Glu280 peptide bond mediated by furin activity is at least partly required for expression of ETA cytotoxicity [2,13] In the present study, our observation that ETA-A associates with hepatic plasma membrane, endosomal and cytosolic fractions isolated from ETA-injected rats is consistent with this view However, our in vivo and in vitro data also support the contention that the furin-mediated conversion of native ETA into ETA-A within hepatic endosomes may represent a minor metabolic fate for the internalized toxin, based on the following: (a) the lack of sensitivity of endosomal ETA-degrading activity to furin inhibitors [hexa-d-arginine (HA)]; and (b) the predominant association of low molecular mass fragments of ETA-A with hepatic endosomes at a late stage of ETA endocytosis (60 post-ETA treatment) Finally, our data suggesting the presence of intact ETA and ETA-A at the cell surface are consistent with the endocytosis of native ETA (major pathway) as well as ETA-A (minor pathway) from the cell surface to early endosomes [28,29] FEBS Journal 277 (2010) 3735–3749 ª 2010 The Authors Journal compilation ª 2010 FEBS 3743 Proteolysis of ETA in rat hepatic endosomes T El Hage et al In the present work, we showed that the endosomal acidic proteolytic activity directed towards the internalized ETA was comparable to that of the cysteine protease cathepsin B and the aspartic acid protease cathepsin D, as indicated by the following observations: (a) the inhibitor profile of the endosomal ETA-degrading activity was very similar to that of cathepsins B and D [30]; and (b) the endosomal activity produced a substrate cleavage pattern that was very similar to that generated with pure cathepsins B or D Interestingly, previous studies have shown that intracellular processing of ETA by a PA-sensitive protease was critical for ETA-induced lymphoproliferation, confirming that one or more intracellular proteases distinct from furin participate in ETA processing within toxin-treated cells [31] Moreover, additional metallo-dependent proteolytic activities (EDTA-sensitive) might act on internalized ETA within endosomes and produce fragments with a molecular mass very close to that of intact ETA All cleavages produced by cathepsins B and D in the ETA toxin are located within ETA-A A major degradation product of ETA results from proteolytic cleavage at Thr396-Cys397 in the C-terminal extremity of domain I or Ib The degradation product contains the entire catalytic ETA-A domain (amino acids 400–613) extended at the N-terminus by the CPV tripeptide, and may represent the main catalytic fragment responsible for the ADP-ribosyltransferase activity identified in vitro after cathepsin treatment Three degradation products (peptides 4a, 4b and 4d) displayed a molecular mass slightly less than that of the native 66 kDa ETA and the unmodified N-terminal ETA sequence, suggesting the removal of the C-terminal residues of ETA encompassing the REDLK sequence However, an antibody that recognizes the REDLK-mediated ER retrieval motif, which is located at the C-terminus of ETA-A, showed immunoreactivity with endosomal ETA-A, suggesting that the REDLK motif was not completely lost from ETA-A within endosomes It has previously been shown that human serum contains a carboxypeptidase activity, suggested to be carboxypeptidase-N, carboxypeptidase-H or carboxypeptidase-M, which removed the C-terminal Lys of ETA and generated a processed form of ETA ending in REDL [15] We have now extended these observations to the endosomal apparatus, and suggest that ETA may undergo C-terminal processing that begins early in the circulating blood, and is continued later within endosomes after entry of the toxin into the cell Western blot analyses of ETA associated with hepatic subcellular fractions under nonreducing conditions showed that the Cys265–Cys287 disulfide bridge was 3744 partially cleaved at the plasma membrane, endosome and cytosol loci Thus, as for the proteolytic cleavage of ETA at the connecting A ⁄ B junction bond, the hepatic ETA-reducing activity may well operate early at the cell surface prior to ETA endocytosis Moreover, the level of ETA reduction within hepatic endosomes was much lower than that of proteolysis, suggesting that the endosomal reductive pathway may represent a minor metabolic fate for the internalized toxin [32] It has been previously suggested that ETA reduction is a two-step process: toxin unfolding that allows access to the Cys265–Cys287 bond is followed by reductive cleavage of the disulfide bond by a protein disulfide isomerase-like enzyme [14] Importantly, toxin unfolding and reducing activities were present in the membrane fraction of toxin-sensitive cells but not in the soluble fraction, suggesting that the cytosol and the endosomal lumen may not be the relevant compartments for such cell-mediated reducing events [14] One endosome-located mechanism that regulates ETA activation and action occurs at the level of organelle acidification [33] First, a low pH has been proposed to be required for the proteolytic cleavage of ETA by furin [34] Thus, whereas furin displays an optimal pH of  for model peptide substrates [35], the proteolysis of ETA by furin is maximal between pH 5.0 and pH 5.5 [34] Moreover, the vacuolar H+-ATPase inhibitor bafilomycin protected mouse L cells from the toxic effects of intact ETA as well as precleaved ETA, suggesting that an acidic environment is required for proteolytic activation of ETA and additional event(s) leading to its cytotoxic effect [33] Finally, it has clearly been shown that endosomal acidity facilitates the binding of ETA to the endosomal membrane of mouse L cells (maximal binding observed at pH 4.0) and ETA-induced pore formation in the lipid bilayer of endosomal vesicles (maximal effect at pH < 6) [8] Our data showing the in vitro proteolysis of ETA by endosomal acidic cathepsins and translocation of the internalized toxin across the endosomal membrane at low pH would be consistent with these prior observations Other studies reported that ETA translocation was strictly dependent on ATP hydrolysis but was not affected by bafilomycin, the H+-ATPase inhibitor [9] These differences may result from the experimental approaches used (the rat liver in vivo model versus cellular in vitro systems) and ⁄ or may be related to differences between hepatocytes and other cell types In vivo [36] and in vitro [37] studies have shown that the normal airway epithelium is highly resistant to P aeruginosa-induced apoptosis Moreover, in airway target cells, ETA induced a wide range of biochemical FEBS Journal 277 (2010) 3735–3749 ª 2010 The Authors Journal compilation ª 2010 FEBS T El Hage et al and morphological changes (early mitochondrial dysfunction) that are not characteristic of apoptosis [23] On the other hand, live P aeruginosa bacteria can induce the apoptotic death of human conjunctiva epithelial Chang cells [38] and J774A.1 macrophages [39] through the type III secretion system Also, ETA-induced human mast cell apoptosis by activation of the caspase-8 and caspase-3 pathways and downregulation of antiapoptotic FLIP proteins has been reported [3] In the present work, we have demonstrated that, after ETA administration, rat hepatic cells undergo in vivo apoptosis through DNA fragmentation, mitochondrial release of cytochrome c, and activation of caspase-9 and caspase-3 By contrast, the receptor caspase-8-dependent pathway did not contribute to ETA-induced apoptosis in rat liver cells in vivo We speculate that, in the cytoplasm of toxin-treated hepatic cells, translocated ETA can effect its cytotoxicity through a dual action, i.e ADP-ribosylation of EF-2 (inhibition of protein synthesis) and mitochondrial alteration (intrinsic apoptotic effect) Both pathways require the translocation of ETA into the cytoplasm of toxin-treated cells On the basis of the reconstitution of the cytotoxic pathways with in vitro cytosol and cell-free mitochondria, our data suggest a direct interaction between ETA and cytosolic EF-2 on the one hand, and the mitochondrial membrane on the other hand However, the potential role (if any) of ADP-ribosylation of EF-2 in the mitochondrial apoptotic response induced by the toxin remains to be determined Finally, we assign an important role to the endosomal acidic cathepsins B and D in increasing the in vitro transfer of the ADP-ribosyl moiety of NAD+ to EF-2 by ETA, but not in the release of cytochrome c from cell-free mitochondria In summary, we found that internalized ETA was rapidly proteolyzed within rat hepatic endosomes by cathepsins B and D, with subsequent ATP-dependent translocation of intact ETA and ETA-A to the cytosol Intact ETA induced ADP-ribosylation of cytosolic EF-2 as well as the mitochondrial release of cytochrome c, both in vivo and in vitro, with the in vitro effects being substantially increased by cathepsin B ⁄ D pretreatment Studies are currently underway to elucidate whether ETA-induced mitochondrial alteration is mediated by the catalytic A-subunit or hydrophobic B-domain of ETA, or whether it involves the dual heterogeneous part of the toxin Use of this approach will provide novel insight(s) into the physiological significance of the endosomal fragments of internalized ETA, which, until now, has remained elusive Proteolysis of ETA in rat hepatic endosomes Experimental procedures Peptides, enzymes, antibodies, protein determination, N-terminal sequencing, enzyme assays, and materials Pseudomonas aeruginosa ETA, DT and bafilomycin-A1 were purchased from Calbiochem Bovine cathepsin D (EC 3.4.23.5, 15 mg)1), bovine cathepsin B (EC 3.4.22.1, recombinant truncated human furin 10 mg)1), (2000 mL)1) and human plasma a2MG were purchased from Sigma Rabbit antibody against Pseudomonas ETA was purchased from Sigma Western blot analysis using the antibody against ETA revealed a strong affinity for ETA with a specificity for the A-subunit Mouse monoclonal antibody directed against rat EEA1 was purchased from Transduction Laboratories Mouse monoclonal antibody directed against rat cytochrome c was purchased from Pharmingen Rabbit polyclonal antibody against human EF-2 and goat polyclonal antibody raised against the C-terminus of human LRP1 were purchased from Santa Cruz Biotechnology Rabbit polyclonal antibody against KX5KDEL, which recognizes the ER retention signal KDEL and binds to various ER-resident proteins, was obtained from S Fuller (EMBL, Heidelberg, Germany) Horseradish peroxidase-conjugated goat anti-(rabbit IgG) or goat anti-(mouse IgG) were from Sigma The protein content of isolated fractions was determined by the method of Lowry et al [40] N-terminal sequence data were obtained by automated Edman degradation with a Procise sequencer (Applied Biosystems, Foster City, CA, USA), equipped with an on-line phenylthiohydantoin amino acid analysis system Quantitative analysis of DNA fragmentation after toxin-induced cell death was analyzed by immunoassay determination of cytoplasmic histone-associated DNA fragments, according to the manufacturer’s protocol (Roche) N-Acetyl-b-d-glucosaminidase was assayed with p-nitrophenyl N-acetyl-b-d-glucosaminide as substrate, according to Touster et al [41] Acid phosphatase was assayed as described by Trouet [42] Caspase-3, caspase-8 and caspase-9 activity was analyzed with a fluorometric assay kit (BioVision, Mountain View, CA, USA) with the respective DEVD-AFC, IETD-AFC and LEHD-AFC substrates Nitrocellulose membranes and the enhanced chemiluminescence detection kit were from Amersham PA, E-64, phenylmethanesulfonyl fluoride and EDTA were from Sigma HA was from Calbiochem All other chemicals were obtained from commercial sources and were of reagent grade Animals and injections In vivo procedures were approved by the institutional committee for use and care of experimental animals Male Sprague-Dawley rats, body weight 180–200 g, were obtained from Charles River France (St Aubin Les Elbeufs, FEBS Journal 277 (2010) 3735–3749 ª 2010 The Authors Journal compilation ª 2010 FEBS 3745 Proteolysis of ETA in rat hepatic endosomes T El Hage et al France) and fed ad libitum Native ETA or DT (15 lg per 100 g body weight) in 0.4 mL of 0.15 m NaCl was injected within s into the penile vein under light anesthesia with ether Rat liver subcellular fractionation cathepsin-treated ETA, according to Uren et al [51] In some experiments, KCl was omitted from the incubation medium to disrupt mitochondria by hypotonic lysis Samples were incubated at 37 °C for various periods (5 to h) and centrifuged for 15 at 30 000 g Supernatants were subjected to reducing SDS ⁄ PAGE followed by western blot analysis with antibody against cytochrome c Subcellular fractionation was performed using established procedures [22,24,25] Following injection of toxins, rats were killed and livers were rapidly removed and minced in ice-cold isotonic homogenization buffer as previously described [22,24,25] Rat liver large granule and cytosolic fractions were isolated by differential centrifugation as previously described [43–46] Plasma membrane was prepared according to the method of Neville [47] as described by Authier et al [43,48,49] The endosomal fraction was isolated by discontinuous sucrose gradient centrifugation and collected at the 0.25–1.0 m sucrose interface [22,24,25] Endosomal fractions revealed no significant enrichment of lysosomal enzyme markers (N-acetyl-b-d-glucosaminidase, relative specific activity = 1.5; acid phosphatase, relative specific activity = 2.2), with the yield of enzymes accounting for < 0.2% of that of the homogenate The recovery of organelle enzyme markers in the nonsedimentable cytosolic fraction was very low, and is in agreement with our previously published biochemical characterizations [43,50] Electrophoresed samples were transferred onto nitrocellulose membranes for 60 at 380 mA in transfer buffer containing 25 mm Tris base and 192 mm glycine The membranes were blocked by a h incubation with 5% skimmed milk in 10 mm Tris ⁄ HCl (pH 7.5), 300 mm NaCl and 0.05% Tween-20 The membranes were then incubated with primary antibody [mouse IgG against rat cytochrome c (diluted : 1000), mouse monoclonal antibody against rat EEA1 (diluted : 1000), rabbit polyclonal IgG against either ETA (diluted : 60 000), KX5KDEL (diluted : 100) [22] or human EF-2 (diluted : 500)] in the above buffer for 16 h at °C The blots were then washed three times with 0.5% skimmed milk in 10 mm Tris ⁄ HCl (pH 7.5), 300 mm NaCl and 0.05% Tween-20 over a period of h at room temperature The bound immunoglobulin was detected with horseradish peroxidase-conjugated goat anti-(rabbit IgG) or goat anti-(mouse IgG) Cell-free proteolysis and translocation of endosome-associated ETA In vitro proteolysis of ETA by hepatic endosomes and proteases Endosomal fractions isolated 30 after the injection of native ETA (15 lg per 100 g body weight) were suspended at mgỈmL)1 in 0.15 m KCl, mm MgCl2 and 25 mm Hepes (pH 7) or 25 mm citrate ⁄ phosphate buffer (pH 5–6) in the presence or absence of 10 mm ATP and 0.01 lm bafilomycin-A1 Samples were incubated at 37 °C for various periods and subjected to reducing SDS ⁄ PAGE followed by western blotting to determine the endosomal content and integrity of ETA and ETA-A To specifically assess the membrane translocation of intact and processed ETA through the endosomal membrane, incubation mixtures were centrifuged for 60 at 100 000 g Pelleted endosomes and supernatants were then subjected to reducing SDS ⁄ PAGE followed by western blot analysis with antibody against ETA The endosomal fraction ( lg) was incubated for varying lengths of time at 37 °C with 10 lg of native ETA in 30 lL of 25 mm citrate ⁄ phosphate buffer (pH 5) or 25 mm Hepes buffer (pH 7) containing mm CaCl2, in the presence or absence of protease inhibitors To determine the integrity of ETA, the proteolytic reaction was stopped by the addition of reducing SDS ⁄ PAGE sample buffer, and this was followed by SDS ⁄ PAGE and western blot analysis For some experiments, ETA (10 lg) was digested in vitro for varying lengths of time with bovine cathepsin B or cathepsin D (5 mL)1Ỉmg)1) in 50 mm citrate ⁄ phosphate buffer (pH 4–6), or human furin (10 mL)1Ỉmg)1) in 50 mm Hepes buffer (pH 7) containing 10 mm CaCl2 and 10 mm dithiothreitol The proteolytic reaction was stopped by the addition of reducing SDS ⁄ PAGE buffer, and this was followed by SDS ⁄ PAGE and Coomassie Brilliant Blue staining or western blot analysis Cell-free translocation of mitochondria-associated cytochrome c A rat liver mitochondrial fraction (large-granule fraction) was isolated by differential centrifugation as previously described [43,46], and then resuspended at 7.5 mgỈmL)1 in 0.15 m KCl, mm MgCl2, mm EDTA and 10 mm Hepes (pH 7.5) in the presence or absence of native or 3746 Immunoblot analysis ETA-catalyzed ADP-ribosylation of cytosolic EF-2 Native ETA (10 lg) was incubated 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proteins Electrostatic interactions can hold cytochrome-c but not Smac ⁄ Diablo to mitochondrial membranes J Biol Chem 280, 2266–2274 FEBS Journal 277 (2010) 3735–3749 ª 2010 The Authors Journal compilation ª 2010 FEBS 3749 ... with an antibody against ETA showed that cathepsins B and D degraded ETA in a pH-dependent manner, with maximal degradation being observed at pH The ETA fragments generated by the pure cathepsins. .. Kinetics of appearance of ETA in hepatic plasma membranes and endosomes after toxin administration Rat hepatic plasma membrane (A) and endosomal fractions (B) were isolated at the indicated times after... act on internalized ETA within endosomes and produce fragments with a molecular mass very close to that of intact ETA All cleavages produced by cathepsins B and D in the ETA toxin are located